Legionela pneumophila, the agent of Legionnaires' disease, exists in many environments such as human alveolar macrophages, fresh water protozoa, and biofilms. Cianciotto, et al., Mol. Biol. Med. 6:409-424 (1989); Dowling, et al., Microbiol. Rev. 56:32-60 (1992); Fields, Trends Genet. 4:286-290 (1996); Horwitz, Curr. Top. Microbiol Immunol. 181:265-282 (1992). To survive, the bacterium must obtain essential nutrients from its environment, and the capacity to sense the availability of a particular nutrient may be important for the regulation of its uptake. Iron is essential for L. pneumophila growth both extracellularly and intracellularly Byrd and Horwitz, J. Clin. Invest. 83:1457-1465 (1989); Feely, et al., J. Clin. Microbiol. 8:320-325 (1978); James, et al., Infect. Immun. 63:4224-4230 (1995); Pine, et al., J. Clin Microbiol. 9:615-626 (1979); Pope, et al., Infect. Immun. 64:629-36 (1996); Reeves, et al., J. Clin Microbiol. 13:688-695 (1981)!, serving as a co-factor for an aconitase, a superoxide dismutase, and other enzymes Hoffman, in Legionella: Proceedings of the 2nd International Symposium, American Society for Microbiology, Washington, D.C., p. 61-67 (Thornsberry, et al. eds., 1984); Mengaud and Horwitz, J. Bacteriol. 175:5666-5676 (1993); Steinman, Mol. Gen. Genet. 232:427-430 (1992)!. L. pneumophila requires 3-13 .mu.M ferric iron for extracellular growth, a level that is relatively high compared to other bacteria. Hoffman, in Legionella: Proceedings of the 2nd International Symposium, American Society for Microbiology, Washington, D.C., p. 61-67 (Thornsberry, et al. eds., 1984); James, et al., Infect. Immun. 63:4224-4230 (1995); Johnson, et al., Infect. Immun. 59:2376-2381 (1991); Mengaud, et al., J. Bacteriol. 175:5666-5676 (1993); Pine, et al., J. Clin Microbiol. 9:615-626 (1979); Reeves, et al., J. Bacteriol. 154:324-329 (1983); Ristroph, et al., J. Clin. Microbiol. 13:115-119 (1981). However, iron within the macrophage host is either bound by transferrin, complexed as ferritin, or sequestered in the labile iron and heme pools. Bridges, in Iron storage and transport, CRC Press, Boca Raton, pp. 297-314 (Ponka, et al. eds., 1990); Crichton and Charloteaux-Wauters, Eur. J. Biochem. 164:485-506 (1987); Muller-Eberhard and Nikkila, Semin. Hematol. 26:86-104 (1989). Thus, the intraphagosomal L. pneumophila likely has evolved strategies for scavenging scarce free-iron or iron bound to cellular chelators. However, the ways in which L. pneumophila obtains iron from its environment remain unknown. High affinity siderophores that are commonly used for Fe.sup.3+ assimilation by other bacteria are reported to be absent from the legionellae. Liles and Cianciotto, Infect Immun. 64:1873-1875 (1996); Reeves, et al., J. Bacteriol. 154:324-329 (1983). Also, this bacterium does not bind and utilize transferrin or lactoferrin. Bortner, et al., Can. J. Microbiol. 35:1048-1051 (1989); Byrd and Horwitz, J. Clin. Invest. 83:1457-1465 (1989); Byrd and Horwitz, J. Clin Invest. 88:1103-1112 (1991); Johnson, et al., Infect. Immun. 59:2376-2381 (1991); Quinn and Weisberg, Curr. Microbiol. 17:111-116 (1989). L. pneumophila does, however, express two internal ferric reductases which likely process internalized Fe.sup.3+. Johnson, et al., Infect. Immun. 59:2376-2381 (1991); Poch and Johnson, Biometals 6:107-114 (1993). Undoubtedly, the legionellae have multiple iron uptake systems, like so many other pathogens. Guerinot, Annu. Rev. Microbiol. 48:743-72 (1994); Otto, et al., Crit. Rev. Microbiol. 18:217-233 (1992); Payne, Trends Microbiol. 1:66-69 (1993); Wooldridge and Williams, FEMS Microbiol Rev. 12:325-348 (1993).
In other bacteria, the factors involved in iron uptake, such as siderophores and transferrin receptors, are regulated by the repressor protein Fur. Bagg and Neilands, Biochemistry 26:5471-5477 (1987); de Lorenzo, et al., J. Bacteriol. 169:2624-2630 (1987); Guerinot, Annu. Rev. Microbiol. 48:743-72 (1994); Litwin, Clin. Microbiol. Rev. 6:137-149 (1993); Tsolis, et al., J. Bacteriol. 177:4628-37 (1995). In the presence of ferrous iron, the Fur dimer binds to promoter regions and represses the transcription of iron acquisition genes. Bagg and Neilands, Biochemistry 26:5471-5477 (1987); Hantke, Mol. Gen. Genet. 182:228-292 (1981). However, when the availability of iron is low, Fur does not bind and the genes are expressed. In addition to the controlling factors involved in iron uptake, Fur represses variety of other virulence determinants, such as exotoxin A in Pseudomonas aeruginosa and a hemolysin in Vibrio cholerae. Litwin, et al., J. Bacteriol. 174:1897-1903 (1992); Prince, et al., J. Bacteriol. 175:2589-2598 (1993).
Recently, the L. pneumophila fur gene was isolated and its predicted amino acid sequence was shown to be 50-60% identical to that of other bacteria. Hickey and Cianciotto, Gene 143:117-121 (1994). Furthermore, the cloned fur encoded a protein able to repress Escherichia coil fiu expression and decrease enterobactin production. These data suggested that L. pneumophila iron acquisition is regulated by Fur using the concentration of iron in the environment as a signal for when to repress gene expression.
A number of pathogens, including both intra- and extracellular parasites, have evolved mechanisms for interacting with hemin and heme-containing compounds. Otto, et al., Crit. Rev. Microbiol., 18:217-233 (1992); Wooldridge and Williams, FEMS Microbiol. Rev. 12:325-348 (1993). This interaction can serve several functions. First, and perhaps most often, it serves as a means for iron acquisition. Strains of Aeromonas spp., Bacteroides fragilis, Bordetella pertussis, Campylobacter jejuni, Escherichia coli, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Neisseria gonorrheae, Neisseria meningitidis, Plesiomonas shigelloides, Porphyromonas gingivalis, Serrati marcescens, Shigella flexneri, Streptococcus pneumoniae, Vibrio cholerae, Vibrio vulnificus, Yersinia enterocolitica, and Yersinia pestis scavenge the iron from hemin and related substances. Daskaleros and Payne, Infect. Immun. 55:1393-1398 (1987); Daskaleros, et al., Infect. Immun. 59:2706-2711 (1991); Genco, et al., Infect. Immun. 62:2885-2892 (994); Lee, J. Med. Microbiol. 34:317-322 (1991); Letoffe, et al., Proc. Natl. Acad. Sci. USA, 91:9876-9880 (1994); Massad, et al., J. Gen. Microbiol. 137:237-241 (1991); Otto, et al., Crit. Rev. Microbiol. 18:217-233 (1992); Tal, et al., Infect. Immun. 61:5401-5405 (1993); Wooldridge and Williams, FEMS Microbiol. Rev. 12:325-348 (1993); Worst, et al., Infect. Immun. 63:4161-4165 (1995). Second, in H. influenzae and P. gingivalis, heme structures are absolutely required as sources for porphyrin rings. Carman, et al. Infect. Immun. 58:4016-4019 (1990); Cope, et al., J. Bacteriol. 177:2644-2653 (1995). Third, hemin binding to a bacterial surface may facilitate infection of eukaryotic cells. For example, in S. flexneri and enteroinvasive E. coli, it enhances invasion of epithelial cells, whereas in Aeromonas salmonicida, hemin interaction with S-layer proteins promotes macrophage association. Daskaleros and Payne, Infect. Immun. 55:1393-1398 (1987); Garduno and Kay, Infect. Immun. 60:4612-4620 (1992); Stugard, et al., Infect. Immun. 57:3534-3539 (1989). Fourth, hemin (heme) serves as a cofactor for intracellular cytochromes and enzymes; e.g., the heme-binding FixL of Rhizobium meliloti is an oxygen-sensing membrane kinase. Monson, et al., Proc. Natl. Acad. Sci. USA 89:4280-4284 (1992). Finally, Y. pestis has an extraordinary capacity to store hemin. Perry, Trends Microbiol. 1:142-147 (1993). In recent years, a number of investigators have turned their attention toward the molecular and genetic bases of hemin binding and utilization. This complex process generally involves the concerted effort of surface/outer membrane receptors and periplasmic/inner membrane transporters and has been characterized as a TonB-dependent uptake event. Bramanti and Holt, J. Bacteriol. 175:7413-7420 (1993); Cope, et al., J. Bacteriol. 177:2644-2653 (1995); Elkins, et al., Infect. Immun. 63:2194-2200 (1995); Hanson and Hansen, Mol. Microbiol. 5:267-278 (1991); Henderson and Payne, J. Bacteriol. 176:3269-3277 (1994); Lewis and Dyer, J. Bacteriol. 177:1299-1306 (1995); Mills and Payne, J. Bacteriol. 177:3004-3009 (1995); Stojiljkovic and Hantke, Mol. Microbiol. 13:719-732 (1994). In S. marcescens, heme acquisition is initiated by an extracellular heme-binding protein. Letoffe, et al., Proc. Natl. Acad. Sci. USA 91:9876-9880 (1994).
The relationship between L. pneumophila and hemin has received very little attention. Although ferric/ferrous iron clearly plays a critical role in extra- and intracellular Legionella growth, Byrd and Horwitz, J. Clin. Invest. 83:1457-1465 (1989); Feeley, et al., J. Clin. Microbiol. 8:320-325 (1978); Gebran, et al., Infect. Immun. 62:564-568 (1994); Quinn and Weisberg, Curr. Microbiol. 17:111-116 (1988); Reeves, et al., J. Clin. Microbiol. 13:688-695 (1981)!, the role of hemin is unclear. Several early studies demonstrated bacterial growth on complex and semi-defined media which contained hemin or hemoglobin supplements. Feeley, et al., J. Clin. Microbiol. 8:320-325 (1978); Pine, et al., J. Clin. Microbiol. 9:615-626 (1979). However, since the heme compounds were easily replaced with ferric salts, it was assumed that they were not required for Legionella growth. Feeley, et al., J. Clin. Microbiol. 8:320-325 (1978). This idea was later confirmed by the development of defined media which completely lacked hemin but supported effective L. pneumophila replication. Reeves, et al., J. Clin. Microbiol. 13:688-695 (1981); Ristroph, et al., J. Clin. Microbiol. 13:115-119 (1981); Warren and Miller, J. Clin. Microbiol. 10:50-55 (1979). Nevertheless, hemin and hemoglobin did enhance L. pneumophila growth on several types of complex media. Current protocols in molecular biology (Ausubel, et al., eds., 1987); Johnson, et al., J. Clin. Microbiol. 15:342-344 (1982). In one study, the growth of seven L. pneumophila strains, representing six serogroups, was stimulated by .gtoreq.100-fold by the addition of hemin to a yeast extract phosphate (YP) medium. Johnson, et al., J. Clin. Microbiol. 15:342-344 (1982). Taken together, these data suggest that hemin can serve as an accessory iron source.